Carcinoma of the cervix is one of the most common malignancies in women, and worldwide it is second only to breast cancer in both incidence and mortality (NIH Consensus Statement (1) Cervical Cancer. 1996.14:1-18). In developed countries in general, and the United States in particular, the wide acceptance of the Pap smear screening program has resulted in dramatic decreases in the incidence of, and mortality from, cervical cancer. However, a recent survey conducted in United States still showed that an estimated 15,700 women were diagnosed with invasive cervical carcinoma, and an estimated 4,900 women died of cervical cancer per annum (NIH Consensus Statement (1) Cervical Cancer. 1996.14:1-18). Moreover, the incidence of adenocarcinorna has tripled in the past two decades (McGonigle K F, and Berek J S. Early stage squamous cell and adenocarcinoma of the cervix. Curr. Opin. Obstet. Gynecol., 1992. 4:109-119), and currently represents up to 25% of all cervical cancers diagnosed (Miller B E, Flax S D, Arheart K, Photopulos G. The presentation of adenocarcinoma of the uterine cervix. Cancer 1993. 72:1281-1285; and Crum C P, Cibas E S, Lee K R. Glandular precursors, adenocarcinomas, and their mimics. In: Pathology of early cervical neoplasia. New York: Churchill Living stone, 1997. 177-240).
Many factors have been implicated in the relatively high incidence of cervical cancer but the inherently high rate of false negatives appears to be one of the major concerns in the current system of screening. The errors have been attributed, in part, to technical problems, but also may well be based primarily on human factors that are not remedied readily by rules and regulations (Kos L G. Cervical (Pap) smear. New directions. Cancer (Phial.), 71 (suppl.); 1993. 1406-1412).
In recent years, the use of new cervical sampling devices (e.g. cytobrushes) have improved the detection of both glandular and squamous neoplasms involving the endocervical canal. However, the increased endocervical cell yields have also created many new patterns of benign and neoplastic endocervical cytology with significant overlapping features. These patterns are unfamiliar to cytologists in routine daily practice. This uncertainty has been reflected in an increase in the cytologic diagnosis of endocervical glandular atypia.
The morphological criteria for a Pap smear diagnosis of AIS and adenocarcinoma have been well described (Ayer B, Pacey F, Greenberg M, Bousfield L. The cytologic diagnosis of adenocarcinoma in situ of the cervix uteri and related lesions. I. Adenocarcinoma in situ. Acta Cytol., 1987. 31:397-411; and Pacey N F. Glandular neoplasms of the uterine cervix. In: Bibbo M, ed. Comprehensive Cytopatholog. Philadelphia: W B Saunders, 1991. 243-255). However, false-negative rates of up to 40 percent in Pap smears from women later found to have AIS and/or invasive endocervical adenocarcinoma have been reported (Crum et al.,1997, 177-240; and Kim H S, Underwood D, Frable W J. Adenocarcinoma in the cervicovaginal Papanicolaou smear: analysis of a 12-year experience. Diagn. Cytopathol., 1991. 7:119-124). The earlier misdiagnosing of AIS/CA was attributed to the presence of low numbers of endocervical glandular cells in the Pap smears, and/or technical artifacts. However, studies have also indicated that human error may play an important role. This includes the incorrect diagnosis of dysplastic glandular cells as exfoliative endometrial cells and/or reactive endocervical cells (Kos et al., 1993; and Lee K R, Manna E A, St. John T. Atypical endocervical glandular cells: accuracy of cytologic diagnosis. Diagn. Cytopathol. 1995. 13:202-208).
In an attempt to clarify the situation, the Bethesda System committee in 1988 established cytologic criteria for atypical glandular cells, and proposed a new diagnostic terminology with a subclassification scheme, in order to aid in patient triage. It was recommended that endocervical glandular abnormalities that exceed reactive change but fall short of invasive adenocarcinoma be reported as atypical glandular cells of undetermined significance (AGUS) (National Cancer Institute Workshop. The revised Bethesda System for reporting cervical/vaginal cytologic diagnosis: report of the 1991 Bethesda Workshop. JAMA, 1992. 267:1892; and Kurman R J, Solomon D. In The Bethesda System for Reporting CervicalNaginal Cytologic Diagnoses. New York: Springer-Verlag, 1994. 64-76). Up to 1.25 million American women receive a diagnosis of AGUS annually (Raab S S, Geisinger K R, Silverman J F, Thomas P A, Stanley M W. Interobserver variability of a Papanicolaou smear diagnosis of atypical glandular cells of undetermined significance. Am. J. Clin. Pathol., 1998. 110:653-659). However, the results of this subclassification have yet to be proven clinically effective.
A broad morphologic spectrum of benign and neoplastic lesions are included in the category of AGUS. These include atypical endocervical repair, endometriosis, microglandular hyperplasia, tubal metaplasia, squamous intraepithelial lesion (SIL) with glandular involvement, and glandular dysplasia, and adenocarcinoma (Lee K R. Atypical glandular cells in cervical smears from women who have undergone cone biopsy. A potential diagnostic pitfall. Acta. Cytol., 1993. 37:705-709; Pacey F, Ayer B, Greenberg M. The cytologic diagnosis of adenocarcinoma in situ of the cervix uteri and related lesions. III. Pitfalls in diagnosis. Acta. Cytol., 1988. 32:325-330; Yahr U, Lee K R. Cytologic findings in microglandular hyperplasia of the cervix. Diagn. Cytopathol. 1991. 7:248-251; Novotny DB, Maygarden S J, Johnson D E, Frable W J. Tubal metaplasia. A frequent potential pitfall in the cytologic diagnosis of endocervical glandular dysplasia on cervical smears. Acta. Cytol., 1992. 36:1-10; Pacey et al., 1991; and Kos L G. Diagnostic cytology and its histopathologic bases. Vol. 1. 4th ed. Philadelphia; J B Lippincott: 1992. 387:452-454).
Several laboratories have conducted follow-up studies in those cases diagnosed as endocervical atypia. The results indicate that approximately 40% of AGUS cases represent high grade SIL, adenocarcinoma in-situ (AIS), or carcinoma, and correspondingly 60% represent benign or insignificant lesions. A surprising range (15-58%) of patients had significant lesions (HSIL, AIS/CA) in follow-up biopsies, after receiving a Pap smear diagnosis of AGUS. Furthermore, only a small fraction of the significant lesions were glandular neoplasms (AIS/CA). Thus, the AGUS diagnosis appears to be a misnomer inasmuch that many AGUS lesions are not glandular at all (Wilbur D C, Mulford D M, Sickel J Z, Atkinson K M. The problem of endocervical atypia: new cytologic presentations of normal endocervical cells. Mod. Pathol., 1994. 38:808; Goff B A., Atanasoff P, Brown E, Muntz H G, Bell D A, Rice L W. Endocervical glandular atypia in Papanicolaou smears. Obstet. Gynecol., 1992. 79:101-104; Lee et al., 1995; Nasu I, Meurer W, Fu Y S. Endocervical glandular atypia and adenocarcinoma: A correlation of cytology and histology., Int. J. Gynecol. Pathol., 1993. 12:208-218; Taylor R, Guerriere J, Nash J D, Henry M R, O""Conner D M. Atypical cervical cytology: Colposcopic follow-up using The Bethesda System, J. Reprod. Med., 1993. 38:443-447; Kennedy A W, Salmieri S S, Wirth S L, Biscotti C V, Tuason L J, Travarca M J. Results of the clinical evaluation of atypical glandular cells of undetermined significance (AGUS) detected on cervical cytology screening. Gynecol. Oncol., 1996. 63:14-18; and Bose S, Kannan V, Kline T S. Abnormal endocervical cells. Really abnormal! Really endocervical! Am. J. Clin. Pathol., 1994. 101:708-713).
There have been many attempts to define cytologic criteria that aid in the diagnosis of glandular lesions of the cervix, with the goal of separating neoplastic glandular cells from reactive changes and squamous neoplasia. Yet, there remains poor agreement among cytopathologists in reclassifying lesions originally diagnosed as AGUS, and in separating clinically significant from benign AGUS lesions (Raab et al., 1998; Novotny et al., 1992; Nasu et al.,1993; Raab S S, Isacson C, Layfield L J, Lenel, J C, Slagel, D D, Thomas P A. Atypical glandular cells of undetermined significance: cytologic criteria to separate clinically significant from benign lesions. Am. J. Clin. Pathol., 1995. 104:574-582; Raab S S, Snider T E, Potts S A, McDaniel H L, Robinson R A, Nelson D L, Sigman J D, Thomas P A. Atypical glandular cells of undetermined significance. Diagnostic accuracy and interobserver variability using select cytologic criteria. Am. J. Clin. Pathol., 1997. 107:299-307; and DiTomasso J P, Ramzy I, Mody D R. Glandular lesions of the cervix.: Validity of cytologic criteria used to differentiate reactive changes, glandular intraepithelial lesions and adenocarcinoma. Acta. Cytol., 1996. 40:1127-1135).
This diagnostic uncertainty has posed a particular dilemma in clinical management decisions, both from a cost-benefit standpoint and a desire not to subject patients to unnecessary invasive procedures. Therefore, there is a need for a useful discriminator of AGUS diagnoses that can separate glandular cells that are atypical due to reactive-reparative changes from cells that are atypical due to dysplasia and carcinoma.
Recently, a novel antigen, termed MN/CA9, has been described. It is a transmembrane glycoprotein that was discovered in the cervical adenocarcinoma cell line, Hela (Zavada J, Zavadova Z, Pastorekova S, Ciampor F, Pastorek J, Zelnik V. Expression of MaTu-MN protein in human tumor cultures and in clinical specimens. Int. J. Cancer 1993. 54:268-274). The gene encoding the MN/CA9 product is unusualxe2x80x94the only homologous functional domain identified to date being a carbonic anhydrase domain. Evidence supporting the role of the MN/CA9 protein in neoplastic progression includes its association with the tumorigenic phenotype in human cell hybrids and neoplastic transformation of mouse 3T3 cells following transfection with MN cDNA (Zavada et al., 1993; and Pastorek J, Pastorekova S, Callebaut I, Morrion J P, Zelnik V, Opavsky R, Zatovicova M, Liao S, Portelle D, Stanbridge E J, Zavada J, Burny A, Kettmann R. Cloning and characterization of M N, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment. Oncogene 1994. 9:2877-2888). A preliminary screen of clinical specimens indicated that expression of MN/CA9 protein is restricted to very few normal tissues, but significant levels of expression were noted in certain malignancies, including cervical neoplasms (Pastorek et al., 1994).
A study of several hundred benign and neoplastic cervical specimens has shown that MN/CA9 is expressed in all cases of AIS and in more than 90% of cervical squamous neoplasms. High levels of MN/CA9 protein expression were frequently observed in the normal-looking endocervical cells in regions adjacent to dysplastic tissues but the normal cervix does not express MN/CA9 protein. In addition, a study of 305 Pap smears has also indicated that the MN/CA9 expression seen in exfoliative cells in Pap smears recapitulates MN/CA9 expression in the corresponding tissue sections of the cervix. Virtually all atypical glandular cells derived from AIS and adenocarcinoma expressed high levels of MN antigen, whereas endocervical cells obtained from benign cervices were negative (Liao S Y, Brewer C, Zavada J, Pastorek J, Pastorekova S, Marietta A, Berman M L, DiSaia P J, Stanbridge E J. Identification of the MN antigen as a diagnostic biomarker of cervical intraepithelial squamous and glandular neoplasia and cervical carcinomas. Am. J. Pathol., 1994. 145: 598-609; Liao S Y, Stanbridge E J. Expression of the MN antigen in cervical Papanicolaou smears is an early diagnostic biomarker of cervical dysplasia. Canc. Epid. Biom. Prev., 1996. 5:549-557).
While these results have shown that expression of the MN/CA9 antigen can indicate AIS and adencarcinoma, they have not enabled reliable distinction between high grade and low grade lesions and benign conditions. Enabling such a distinction is critical to meaningful clinical diagnosis. The deadly harm caused by false negatives, and the anguish and expense caused by false positives demands a more reliable clinical procedure.
The present invention fulfills the need for a more accurate diagnosis of AGUS than the current routine conventional Pap smear screening. The invention provides new insight into the distribution of MN/CA9 antigen on atypical and/or normal cells on AGUS-diagnosed Pap smear specimens and serves as a clinical roadmap enabling MN/CA9 antigen to at last serve as a powerful diagnostic biomarker for cancerous or pre-cancerous conditions.
Specifically, the present invention for the first time provides a reliable comprehensive method to determine the presence of cancerous or pre-cancerous cervical lesions from Pap smear cells that have been cytologically diagnosed as atypical glandular cells of undetermined significance (AGUS) under the Bethesda System of terminology. The AGUS-diagnosed Pap smear cells are subjected to a procedure that detects expression of MN/CA9 antigen, such as immunohistochemistry or in situ cytohybridization. AGUS-diagnosed Pap smears typically lack readily ascertainable dysplastic cells, but do include atypical and normal endocervical cells. It is the distribution of MN/CA9 antigen observed on atypical or normal cells that is used to diagnose the presence of significant or low grade lesions. More specifically, significant lesions, including adenocarcinoma, invasive carcinoma (CA), or high grade squamous intraepithelial lesions (HSIL) are diagnosed when MN/CA9 antigen is observed on atypical cells. Low grade lesions, including low grade squamous intraepithelial lesions (LSIL) or atypia, are diagnosed when MN/CA9 antigen is absent from atypical cells but is present on normal endocervical cells. A benign condition is diagnosed when MN/CA9 is absent from atypical and normal endocervical cells.
In preferred versions of the present invention, not only the presence, but the nature of, a significant lesion is diagnosed whenever MN/CA9 antigen is observed on atypical cells. In one embodiment, the presence of adenocarcinoma, including adenocarcinoma in situ (AIS) and invasive adenocarcinoma, is diagnosed when MN/CA9 antigen is detected on atypical cells in a columnar or honeycomb configuration. In another embodiment, the presence of HSIL is diagnosed when MN/CA9 antigen is detected on atypical cells in a tight cluster.
Accordingly, the methods of the present invention overcome problems of uncertainty and false negatives associated with the AGUS diagnosis for Pap smear specimens. In addition, the method discriminates among atypical Pap smears that are associated with significant lesions, low grade lesions or a benign condition.